Fiabilidad de un examen clínico objetivo estructurado para predecir la futura competencia profesional de estudiantes de medicina / Reliability of an objective structured clinical examination to assess the future professional competence of medical students

Introducción: Cuando con los resultados de un examen clínico objetivo estructurado (ECOE) se decide sobre la futura competencia profesional de estudiantes de medicina, la fiabilidad de dicha prueba debe adecuarse a esta finalidad. Objetivo: Calcular la fiabilidad (alfa de Cronbach) de una serie de ECOE y su relación con la duración, número de participantes, estaciones, ítems y evaluadores. Sujetos y métodos: Se analizan 14 ECOE realizados a 2.995 estudiantes de cuarto y quinto curso de la Facultad de Medicina de Granada desde 2004 a 2013. Resultados: La fiabilidad fue ≥ 0,70 en el 92,84% de los ECOE. También fue significativamente ≥ 0,70 cuando la duración total fue ≥ 60 minutos (p = 0,042), el número de estaciones ≥ 10 (p = 0019), el número de ítems ≥ 50 (p = 0,018) y el número de evaluadores ≥ 6 (p = 0,018). No se observaron diferencias con el número de estudiantes ni con las opciones al ítem utilizadas. Conclusiones: Los ECOE cuyos resultados se utilicen para aprobar asignaturas de la carrera de medicina deben tener una fiabilidad ≥ 0,70. Para alcanzar dicha fiabilidad o mayor, el formato debe constar de al menos 10 estaciones, durar ≥ 60 minutos, tener ≥ 50 ítems y ≥ 6 evaluadores

Introduction: When the future professional competence of medical students is decided based on results of an objective structured clinical examination (OSCE), the reliability of this test should be adequate to this purpose. Aim: To calculate the reliability (Cronbach's alpha) of each one of OSCEs we performed and its relationship with the duration, number of participants, stations, items and evaluators. Subjects and methods: Fourteen OSCE tests performed to 2995 medical students of 4th and 5th year of the Faculty of Medicine of Granada between 2004 to 2013 were analyzed. Results: The reliability was ≥ 0.70 in 92.84% of the OSCEs. It was also significant ≥ 0.70 with a total duration ≥ 60 minutes (p = 0.042), and a number of stations ≥ 10 (p = 0.019), a number of items ≥ 50 (p = 0.018) and a number of evaluators ≥ 6 (p = 0.018). No differences with the number of students, neither with the options to the item were observed. Conclusions: The OSCEs carried out in centers which results are used to approve subjects of the medical career, must have a reliability ≥ 0.70. To achieve this reliability or greater, the format should consist of at least: 10 stations, a duration ≥ 60 minutes, and having ≥ 50 items and ≥ 6 evaluators

Acute Stress and Anxiety in Medical Residents on the Emergency Department Duty.

The objectives of this longitudinal study were to compare salivary cortisol release patterns in medical residents and their self-perceived anxiety levels between a regular working day and a day when on call in the emergency department (ED-duty day) and to determine any differences in cortisol release pattern as a function of years of residency or sex. The study included 35 residents (physicians-in-training) of the Granada University Hospital, Granada, Spain. Acute stress was measured on a regular working day and an ED-duty day, evaluating anxiety-state with the Spanish version of the State-Trait Anxiety Inventory. Physiological stress assessment was based on salivary cortisol levels. Cortisol release concentrations were higher on an ED-duty day than on a regular working day, with a significantly increased area under the curve (AUC) (p < 0.006). This difference slightly attenuated with longer residency experience. No gender difference in anxiety levels was observed (p < 0.001). According to these findings, the hypothalamic-pituitary-adrenal axis activity and anxiety levels of medical residents are higher on an ED-duty day than on a regular working day.

Furan and p-xylene as candidate biomarkers for prostate cancer.

BACKGROUND: Prostate cancer (PCa) is the most frequently diagnosed noncutaneous malignant tumor among males in the Western world. Prostate-specific antigen has been considered the most important biomarker for PCa detection; however, it lacks specificity, leading to the search for alternative biomarkers. Volatile organic compounds (VOCs) are released during cell metabolism and can be found in exhaled breath, urine, and other fluids. VOCs have been used in the diagnosis of lung, breast, ovarian, and colorectal cancers, among others. The objective of this study was to identify urinary VOCs that may be sensitive and specific biomarkers for PCa. METHODS: The study included 29 patients with PCa and 21 with benign prostatic hyperplasia. Urine samples were obtained from all participants before and after prostate massage. VOCs were identified by gas chromatography-mass spectrometry. IBM SPSS Statistics v.20 was used for statistical analysis. Sample normality and homogeneity of variances were studied and, according to the distribution normality, ANOVA or the Kruskal-Wallis test was applied to evaluate significant differences between groups. The Pearson test was used to establish correlations. RESULTS: Fifty-seven VOCs were identified. Samples gathered before prostate massage showed significant between-group differences in urinary levels of furan (P≤ 0.001), 2-ethylhexanol (P = 0.032), 3,5-dimethylbenzaldehyde (P = 0.027), santolin triene (P = 0.032), and 2,6-dimethyl-7-octen-2-ol (P = 0.003). Samples gathered after prostate massage showed significant differences in urinary levels of furan (P≤ 0.001), 3- methylphenol (P = 0.014), p-xylene (P = 0.002), phenol (P≤ 0.001), and 2-butanone (P = 0.001). CONCLUSIONS: Significant differences between PCa and BPH patients were found in urinary levels of certain VOCs both before and after prostate massage, supporting the proposal that VOCs may serve as PCa-specific biomarkers.

Zygosaccharomyces rouxii strains CECT 11923 and Z. rouxii CECT 10425: Two new putative hybrids?

Based on IGS-PCR RFLP polymorphism, we previously detected two Z. rouxii strains (CECT 11923 and CECT 10425) that clustered with hybrid strains (NCYC 1682, NCYC 3060 and NCYC 3061). Given the recently recognized important industrial role of hybrids, their detection is very useful. Based on the IGS1 rDNA region alignment of hybrid strains and the Z. rouxii CECT 11923 and CECT 10425, in this work, we developed a pair of Zygosaccharomyces hybrid-specific primers, HibZF/HibZR. Positive amplicons were only obtained in the Zygosaccharomyces spp. hybrids included in this study and the CECT 11923 and CECT 10425 strains analyzed here. In the present study, we applied molecular tools to highlight the nature of these strains; they are quite different from each other as well as from Z. rouxii type strain. Based on the presence of two heterologous copies of nuclear-encoded genes (SOD2 and HIS3), the sequences of divergent 5.8S-ITS rDNA, D1/D2 26S rDNA copies and, the amplification with species-specific primer for Z. rouxii and Z. pseudorouxii, we hypothesize that the CECT 11923 strain might be a hybrid strain. Whereas, CECT 10425, the sequence analysis of 5.8S-ITS rDNA and D1/D2 26S rDNA copies presented 99-100% sequence identity with Zygosaccharomyces sp. NBRC 10669 (LN849119.1) and Z. sapae ABT 301T. Nevertheless, we discard that it could be a Z. sapae strain based on the results obtained in this study. Namely, the amplification with hybrid-specific primer designed in this study, the number of divergent copies of HIS3 (2), the fact that it only possesses one SOD2 gene and the amplification with species-specific primer for Z. pseudorouxii, therefore it could be a new species or a hybrid strain.

Development of an affordable typing method for Meyerozyma guilliermondii using microsatellite markers.

Despite previously published methods, there is still a lack of rapid and affordable methods for genotyping the Meyerozyma guilliermondii yeast species. The development of microsatellite markers is a useful genotyping method in several yeast species. Using the Tandem Repeat Finder Software, a total of 19 microsatellite motifs (di-, tri-, and tetra- repetition) were found in silico in seven of the nine scaffolds published so far. Primer pairs were designed for all of them, although only four were used in this work. All microsatellite amplifications showed size polymorphism, and the results were identical when repeated. The combination of three microsatellite markers (sc15F/R, sc32 F/R and sc72 F/R) produced a different pattern for each of the Type Culture Collection strains of M. guilliermondii used to optimize the method. The three primer pairs can be used in the same PCR reaction, which reduces costs, in tandem with the fluorescent labeling of only the forward primer in each primer pair. Microsatellite typing was applied on 40 more M. guilliermondii strains. The results showed that no pattern is repeated between the different environmental niches. Four M. guilliermondii strains were only amplified with primer pair sc32 F/R, and subsequently identified as Meyerozyma caribbica by Taq I-RFLP of the 5.8S ITS rDNA. Most out-group species gave negative results even for physiologically similarly species such as Debaryomyces hansenii. The microsatellite markers used in this work were stable over time, which enables their use as a traceability tool.

Assessment of the Factors Contributing to the Growth or Spoilage of Meyerozyma guilliermondii in Organic Yogurt: Comparison of Methods for Strain Differentiation.

In this work we analyze the spoiling potential of Meyerozyma guilliermondii in yogurt. The analysis was based on contaminated samples sent to us by an industrial laboratory over two years. All the plain and fruit yogurt packages were heavily contaminated by yeasts, but only the last ones, containing fermentable sugars besides lactose, were spoiled by gas swelling. These strains were unable to grow and ferment lactose (as the type strain); they did grow on lactate plus galactose, fermented glucose and sucrose, and galactose (weakly), but did not compete with lactic acid bacteria for lactose. This enables them to grow in any yogurt, although only those with added jam were spoiled due to the fermentation of the fruit sugars. Fermentation, but not growth, was strongly inhibited at 8 °C. In consequence, in plain yogurt as well as in any yogurt maintained at low temperature, yeast contamination would not be detected by the consumer. The risk could be enhanced because the species has been proposed for biological control of fungal infections in organic agriculture. The combination of the IGS PCR-RFLP (amplification of the intergenic spacer region of rDNA followed by restriction fragment length polymorphism analysis) method and mitochondrial DNA-RFLP makes a good tool to trace and control the contamination by M. guilliermondii.

Development of species-specific primers for rapid identification of Debaryomyces hansenii.

In this work, we developed a specific PCR assay for Debaryomyces hansenii strains that uses a putative homologous PAD1 region (729 bp) present in this yeast species as a target. The amplification of this sequence with the D. hansenii specific primer pair (DhPADF/DhPADR) was found to be a rapid, specific and an affordable method enabling identification of D. hansenii from other yeast strains. Primers were tested in almost 100 strains, 49 strains from Type Culture Collection belonging to the genus Debaryomyces and to other yeast species commonly found in foods or related genera. These primers were able to discriminate between closely related species of Debaryomyces, such as Debaryomyces fabryi and Debaryomyces subglobosus, with a 100% detection rate for D. hansenii. Also, the method was tested in 45 strains from different foods. Results confirmed the specificity of the PCR method and detected two earlier misidentifications of D. hansenii strains obtained by RFLP analysis of the 5.8S ITS rDNA region. Subsequently we confirmed by sequencing the D1/D2 domain of 26S rDNA that these strains belonged to D. fabryi. We call attention in this work to the fact that the RFLPs of the 5.8S ITS rDNA profiles of D. hansenii, D. fabryi and D. subglobosus are the same and this technique will thus lead to incorrect identifications.

Quantitative analysis of morphological changes in yeast colonies growing on solid medium: the eccentricity and Fourier indices.

The colony shape of four yeast species growing on agar medium was measured for 116 days by image analysis. Initially, all the colonies are circular, with regular edges. The loss of circularity can be quantitatively estimated by the eccentricity index, Ei , calculated as the ratio between their orthogonal vertical and horizontal diameters. Ei can increase from 1 (complete circularity) to a maximum of 1.17-1.30, depending on the species. One colony inhibits its neighbour only when it has reached a threshold area. Then, Ei of the inhibited colony increases proportionally to the area of the inhibitory colony. The initial distance between colonies affects those threshold values but not the proportionality, Ei /area; this inhibition affects the shape but not the total surface of the colony. The appearance of irregularities in the edges is associated, in all the species, not with age but with nutrient exhaustion. The edge irregularity can be quantified by the Fourier index, Fi , calculated by the minimum number of Fourier coefficients that are needed to describe the colony contour with 99% fitness. An ad hoc function has been developed in Matlab v. 7.0 to automate the computation of the Fourier coefficients. In young colonies, Fi has a value between 2 (circumference) and 3 (ellipse). These values are maintained in mature colonies of Debaryomyces, but can reach values up to 14 in Saccharomyces. All the species studied showed the inhibition of growth in facing colony edges, but only three species showed edge irregularities associated with substrate exhaustion.

Kinetics of sugars consumption and ethanol inhibition in carob pulp fermentation by Saccharomyces cerevisiae in batch and fed-batch cultures.

The waste materials from the carob processing industry are a potential resource for second-generation bioethanol production. These by-products are small carob kibbles with a high content of soluble sugars (45-50%). Batch and fed-batch Saccharomyces cerevisiae fermentations of high density sugar from carob pods were analyzed in terms of the kinetics of sugars consumption and ethanol inhibition. In all the batch runs, 90-95% of the total sugar was consumed and transformed into ethanol with a yield close to the theoretical maximum (0.47-0.50 g/g), and a final ethanol concentration of 100-110 g/l. In fed-batch runs, fresh carob extract was added when glucose had been consumed. This addition and the subsequent decrease of ethanol concentrations by dilution increased the final ethanol production up to 130 g/l. It seems that invertase activity and yeast tolerance to ethanol are the main factors to be controlled in carob fermentations. The efficiency of highly concentrated carob fermentation makes it a very promising process for use in a second-generation ethanol biorefinery.

Strain typing of Zygosaccharomyces yeast species using a single molecular method based on polymorphism of the intergenic spacer region (IGS).

Unlike previously reported methods that need a combination of several typing techniques, we have developed a single method for strain typing of the Zygosaccharomyces bailii, Z. mellis and Z. rouxii spoilage species. Strains belonging to other species have also been included for comparison. We have demonstrated that the IGS-PCR RFLP method has a high discriminative power. Considering the three endonucleases used in this work, we have obtained a variability of 100% for Z. mellis and Z. rouxii strains and up to 70% for Z. bailii. We have also detected two misidentified Z. mellis strains (CBS 711 and CBS 7412) which have RFLP patterns with a set of bands characteristic of Z. rouxii strains. Sequencing of 26S rDNA D1/D2 domains and the 5.8-ITS rDNA region confirmed these strains as Z. rouxii. The method also groups three certified hybrid strains of Zygosaccharomyces in a separate cluster.

La formación de pre-grado de los médicos en España: situación actual y expectativas de futuro / Planning of health care professionals in Spain. Pre-graduate training of doctors in Spain. Current situation and expectations for the future

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Free amino acid concentrations in vitreous humor and cerebrospinal fluid in relation to the cause of death and postmortem interval.

We studied free amino acids in vitreous humor and cerebrospinal fluid from 58 cadavers in the course of routine medicolegal autopsies in the city of Granada. The main objective was to establish whether free amino acids contents in these fluids were related with the cause of death, postmortem interval, and severity of the classic signs of asphyxia. The amino acids (aspartic acid, glutamic acid, serine, glutamine, glycine/threonine/histidine, citruline, arginine, alanine, taurine, GABA, tirosine, valine, methionine, isoleucine, phenylalanine/tryptophan, leucine, and lysine) were quantified by high performance liquid chromatography. There were no statistically significant differences in amino acids concentrations in vitreous humor when the different causes of death were considered. Our results did not show any statistically significant relationship when asphyxial score was plotted against the vitreous content of each amino acid. A statistically significant increase with postmortem interval was observed in vitreous taurine (r = 0.3191, p = 0.01461), glutamate (r = 0.4323, p = 0.0007) and particularly in aspartate (r = 0.4508, p = 0.0003).

Four new Candida cretensis strains isolated from Spanish fermented sausages (chorizo): taxonomic and phylogenetic implications.

Four yeast strains were isolated from Spanish traditional fermented sausages (chorizo) spoiled by gas production. Using the classical identification procedures, they were identified as Debaryomyces hansenii. However, they fermented galactose and did not produce positive results in Debaryomyces differential medium (DDM), a growth medium highly specific for this species. Phylogenetic analysis showed identical sequences for the D1/D2 domain of the 26S rRNA gene and almost identical sequences for the 5.8S-ITS region with those of the recently described yeast species Candida cretensis. This result was confirmed by sequencing the gene encoding actin of the type and the new strains. Candida cretensis is a new species included in the so-called Candida kruisii clade that was described from a single strain, isolated from a decaying mushroom in Crete, Greece. The discovery of new strains of C. cretensis in fermented food expands its physiological and ecological diversity. With the description of these new strains isolated from food, three groups of strains can be distinguished within C. cretensis according to the restriction patterns of the intergenic spacer rRNA gene region and on the basis of some physiological properties that are of ecological relevance.

PCR-RFLP analysis of the IGS region of rDNA: a useful tool for the practical discrimination between species of the genus Debaryomyces.

The amplification by PCR of the intergenic spacer region (IGS) of rDNA followed by restriction fragment length polymorphism (RFLP) analysis was evaluated as a potential method for discriminating the 16 species belonging to the genus Debaryomyces. The digestion of this region with some or all the enzymes used in this study (HapII, HhaI and MboI) produced species-specific patterns that permitted differentiation of the species in the genus. With the exception of Debaryomyces vanrijiae, the technique was also efficient for distinguishing the varieties in the species Debaryomyces hansenii (var. hansenii, var. fabryi), Debaryomyces occidentalis (var. occidentalis, var. persoonii) and Debaryomyces polymorphus (var. africanus, var. polymorphus), respectively. PCR-RFLP analysis of the IGS region of rDNA is proposed as a clear and reproducible technique for the practical discrimination of species of the yeast genus Debaryomyces.

Hyperosmotic stress induces metacaspase- and mitochondria-dependent apoptosis in Saccharomyces cerevisiae.

During the last years, several reports described an apoptosis-like programmed cell death process in yeast in response to different environmental aggressions. Here, evidence is presented that hyperosmotic stress caused by high glucose or sorbitol concentrations in culture medium induces in Saccharomyces cerevisiae a cell death process accompanied by morphological and biochemical indicators of apoptotic programmed cell death, namely chromatin condensation along the nuclear envelope, mitochondrial swelling and reduction of cristae number, production of reactive oxygen species and DNA strand breaks, with maintenance of plasma membrane integrity. Disruption of AIF1 had no effect on cell survival, but lack of Yca1p drastically reduced metacaspase activation and decreased cell death indicating that this death process was associated to activation of this protease. Supporting the involvement of mitochondria and cytochrome c in caspase activation, the mutant strains cyc1Deltacyc7Delta and cyc3Delta, both lacking mature cytochrome c, displayed a decrease in caspase activation associated to increased cell survival when exposed to hyperosmotic stress. These findings indicate that hyperosmotic stress triggers S. cerevisiae into an apoptosis-like programmed cell death that is mediated by a caspase-dependent mitochondrial pathway partially dependent on cytochrome c.

A beta-glucuronidase-based agar medium for the differential detection of the yeast Debaryomyces hansenii from foods.

A selective and differential solid medium, Debaryomyces differential medium (DDM), was used for the isolation of Debaryomyces hansenii. This medium is formulated to allow detection of the beta-glucuronidase enzyme using the chromogenic substrate magenta-glucuro.CHA (5Br-6Cl-3indolyl-beta-D-glucuronide, cyclohexylammonium salt). Of the more than 120 microorganisms tested, including yeasts, bacteria, and a filamentous fungus, only D. hansenii produced violet colonies, thus permitting its easy discrimination from other organisms. When quality assessment tests were performed, optimal productivity and selectivity were obtained for D. hansenii. The medium was also satisfactory when used to test naturally contaminated food products.

Differential detection of Debaryomyces hansenii isolated from intermediate-moisture foods by PCR-RFLP of the IGS region of rDNA.

The amplification by PCR of the Intergenic Spacer region (IGS) of rDNA followed by Restriction Fragment Length Polymorphism (RFLP) analysis was evaluated as a potential method for the identification of Debaryomyces hansenii among other yeast species that frequently contaminate Intermediate-Moisture Foods (IMFs). For a first rapid differentiation at the species level, the determination of the IGS-PCR fragment size was found to be a useful approach. The digestion of this region with the enzymes HhaI, HapII and MboI resulted in specific patterns that permit the identification of D. hansenii among other yeast species. This method also permitted the discrimination between the D. hansenii varieties (var. hansenii and var. fabryi) as well as the differentiation of D. hansenii from other species of the genus, such as Debaryomyces pseudopolymorphus or Debaryomyces polymorphus var. polymorphus. The IGS-PCR RFLP method was assayed for the differential detection of D. hansenii in contaminated or spoiled IMF products and compared with traditional identification procedures, resulting in a 100% detection rate for D. hansenii.